The nucleotide and corresponding amino acid sequences are reported for a novel class of mammalian lipoxygenase proteins. The novel lipoxygenase encoding polynucleotides were obtained from human gene trap clones and human cDNA libraries.

The present invention relates to the discovery, identification, and characterization of novel human polynucleotides that encode proteins that share sequence similarity with lipoxygenases. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that lack the disclosed genes or over-express the disclosed genes, or antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed polynucleotides that can be used for diagnosis, drug screening, clinical trial monitoring, or the treatment of physiological or behavioral disorders.
Lipoxygenases are enzymes that mediate the oxidation of lipid substrates. As such, lipoxygenases are involved in the synthesis of leukotrienes. Leukotrienes influence a variety of biological processes, and can serve as, inter alia, potent chemotactic agents, and mediators of inflammation, smooth muscle contraction, etc. Accordingly, lipoxygenases represent a key target for the regulation of a variety of biological pathways and conditions.
The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human lipoxygenase proteins, and the corresponding amino acid sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal and plant lipoxygenase proteins. As such, the novel genes represent a new class of lipoxygenase proteins with a range of homologues and orthologs that transcend a broad range of phyla and species.
The invention comprises (a) polypeptides with SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28; (b) homologues and allelic variants of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28; (c) fragments of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 of any size, for example, from 4 amino acids to less than the full-length of a polypeptide of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 and any number between; (d) fragments of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 that correspond to a functional domain (for example, a catalytic domain, a signal sequence, a ligand binding domain, a regulatory domain, etc.); (e) fusion proteins comprising a polypeptide sequence of any one of (a) through (d); (f) mutant polypeptides (including engineered and naturally occurring mutants) comprising a polypeptide sequence of any one of (a) through (d), including, but not limited to, deletion mutants, insertion mutants, substitution mutants, and mutant polypeptides in which all or a part of at least one of the domains is deleted or altered (e.g., a mutant of the active site with altered substrate specificity).
The invention further comprises (g) polynucleotides with SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29; (h) polynucleotides encoding any one of the polypeptides of the invention including, but not limited to, polypeptides specifically described in (a) through (f) above; (i) polynucleotides capable of hybridizing to a second polynucleotide that is complementary to a polynucleotide described in (g) and/or (h) above under conditions of low, medium, or high stringency; (j) oligonucleotides corresponding to a segment of a polynucleotide described in (g) through (i) above and such oligonucleotides having any size from 2 nucleotides through less than the full-length polynucleotide and any length inbetween.
In certain embodiments, the novel human nucleic acid sequences described herein, encode proteins/open reading frames (ORFs) of 711, 489, 556, 334, 615, 460, 291, 69, 139, 195, 110, 867, 645, and 771 amino acids in length (see SEQ ID NOS:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 respectively).
The invention further comprises antibodies to any one of the polypeptides or polynucleotides of the invention. The invention also comprises host cells that are engineered to contain and/or express any one of the polynucleotides and/or polypeptides of the invention.
The invention also comprises agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof that compete with native NHP, and antibodies. The invention further comprises nucleotide sequences that can be used to inhibit the expression of the described NHPs (e.g., antisense and ribozyme oligonucleotides and/or polynucleotides, and gene or regulatory sequence replacement constructs) or to enhance the expression of the described NHP genes (e.g., expression constructs that place the described gene under the control of a strong promoter system), and transgenic animals that express a NHP transgene, or xe2x80x9cknock-outsxe2x80x9d (which can be conditional) that do not express functional NHP.
Further, the present invention also relates to methods for identifying compounds that modulate, i.e., act as agonists or antagonists of, NHP expression and/or NHP product activity that utilize purified preparations of the described NHPs and/or NHP product, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.
The Sequence Listing provides the sequences of 14 lipoxygenase-like ORFs that are encoded by the described NHP polynucleotides (SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29) and the amino acid sequences (SEQ ID NOS:2, 4, 6, 5 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28) encoded thereby.
Lipoxygenases oxidize, or oxygenate, lipids to produce leukotrienes. Depending on the leukotriene synthesized, a wide variety of biological functions can be affected. Typically, leukotrienes will bind cognate receptors an trigger a biological effect (such as, for example, signal transduction). Interfering with lipoxygenase activity ultimately effects leukotriene production and downstream leukotriene-mediated processes. Alternatively, enhancing lipoxygenase activity in vivo, can boost the effects/activity levels the corresponding biological processes. Various lipoxygenase activities can be found in a variety of cells and tissues in both animals and plants. Three predominant types of lipoxygenases include the 5-, 12-, and 15-lipoxygenases, and each type of lipoxygenase can have additional forms depending upon the tissues or cells in which they are expressed.
The 5-, 12-, and 15-lipoxygenases, and the leukotrienes they produce, have been implicated with a variety of diseases and disorders. Given that leukotrienes can modulate inflammatory reactions, they have been associated with a spectrum of mammalian diseases including, but not limited to, asthma, eye diseases, anaphylaxis, lung disease, hematological disorders, infectious diseases, granulomatosis, abscess, pacreatitis, prostatitis, hepatitis, atherosclerosis, heart disease, graft rejection, thrombosis, restenosis, ulcers, kidney disease, hypertension, dermatoses, cramping, autoimmune disorders (lupus, scleroderma, Crohn""s disease, rheumatoid arthritis, etc.), granulomatosis, hyperproliferative diseases, cancer, nausea, headache, metastases, inflammatory bowel disorder, allergy, cancer, arthritis, eczema, melanoma, erythema, acne, psoriasis, shingles, infectious disease, and diabetes. Accordingly, one embodiment of the present invention are processes for identifying compounds useful for the treatment of one or more of the above diseases and disorders that include the use of one or more of the described lipoxygenase-like genes, proteins, or a novel portion thereof.
Given the biological importance of lipoxygenases, the genes encoding such proteins (and the proteins encoded thereby as well as inhibitors thereof) have been subjected to intense scientific/commercial scrutiny (see, for example, U.S. Pat. Nos. 5,036,105, 5,162,365, 5,504,097, 5,066,679, 5,830,453, 4,761,403, 5,589,506, 5,026,729, and 5,861,268) (all of which are herein incorporated by reference in their entirety).
The presently described NHPs share significant similarity with previously described human lipoxygenases. Expression studies using RT-PCR detect NHP transcripts in, inter alia, neural tissue (i.e., brain, spinal cord, etc.), skin, testis, prostate, adrenal gland, cervix, salivary gland, pancreas, heart, lymphoid cells (lymph node, spleen, thymus), and mammary glands. Northern analysis showed a predominant signal in testis, with less predominant, but longer, transcripts detectable in testis, lymph node, and spinal cord. A full length cDNA of a NHP coding region (with 5xe2x80x2 and 3xe2x80x2 extensions) was isolated from a human brain cDNA library (Edge BioSystems, Gaithersburg, Md.) and sequenced (SEQ ID NO: 29). A possible murine ortholog of the described NHPs is predominantly expressed in skin (Kinzig et al., 1999, Genomics 58:158-164).
The invention encompasses the use of the described NHP nucleotides, NHPs and peptides, as well as antibodies, preferably monoclonal antibodies, or binding fragments, domains, or fusion proteins thereof, or anti-idiotypic variants derived therefrom, that bind NHPs, other antagonists that inhibit binding activity or expression, or agonists that activate NHP activity or increase NHP expression, in the diagnosis and/or treatment of disease.
In particular, the invention described in the subsections below encompasses NHP polypeptides or peptides corresponding to functional domains of NHPs, mutated, truncated or deleted NHPs (e.g., NHPs missing one or more functional domains or portions thereof), NHP fusion proteins (e.g., a NHP or a functional domain of a NHP fused to an unrelated protein or peptide such as an immunoglobulin constant region, i.e., IgFc), nucleotide sequences encoding such products, and host cell expression systems that can produce such NHP products.
The invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of the NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote expression of NHP (e.g., expression constructs in which NHP coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.). The invention also relates to as host cells and animals genetically engineered to express the NHPs (or mutant variants thereof) or to inhibit or xe2x80x9cknock-outxe2x80x9d expression of an animal homolog of an endogenous NHP gene.
The NHPs or peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body.
The use of engineered host cells and/or animals offers an advantage in that such systems allow for both the identification of compounds that interact with an NHP, and also provide information regarding the biological significance of the NHP.
Finally, NHP products (especially soluble derivatives such as peptides corresponding to the NHP), and NHP fusion protein products (such as NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate signal transduction which may act on downstream targets in a NHP-associated leukotriene pathway) can be used to directly treat diseases or disorders.
Nucleotide constructs encoding such NHP products can be delivered to host cells that subsequently express the products in vivo; these genetically engineered cells function as xe2x80x9cbioreactorsxe2x80x9d in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotide constructs encoding functional NHPs, mutant NHPs, as well as antisense and ribozyme molecules can also be used in xe2x80x9cgene therapyxe2x80x9d approaches for the modulation of NHP expression. Thus, the invention also encompasses pharmaceutical formulations and methods for treating biological disorders.

Lipoxygenase proteins and polynucleotides encoding the same


Compositions enriched with natural phyto-oestrogens or analogues thereof selected from Genistein, Diadzein, Formononectin and Biochanin A. These compositions may be used as food additives, tablets or capsules for promoting health in cases of cancer, pre-menstrual syndrome, menopause or hypercholesterolaemia.

This invention relates to natural products containing phyto-oestrogens, or phyto-oestrogen metabolites, which have various beneficial physiological effects in man, and which have a variety of uses, such as to promote good health and as a dietary additive, for example.
The particular product in accordance with the invention is an extract of certain plants with the particular purpose of enrichment for phyto-oestrogens, both in their natural state and their closely related derivatives and metabolites.
Plants which are used as foodstuffs or medicinal herbs contain a wide variety of chemicals which are assimilated into the body following ingestion. Some of these chemicals are important nutrients for man and animals (e.g. fats, carbohydrates, proteins, vitamins, minerals) while others have none, or little or no known nutritional value. The phyto-oestrogens hitherto have fallen into this latter category of no known nutritional value.
There are 3 principal classes of phyto-oestrogens, viz. isoflavones, lignans, and coumestans. The isoflavones are thought to have a broad range of biological functions in plants, although these are poorly understood. However, two particular functions are recognisedxe2x80x94(a) as phyto-alexin or stressor chemicals which are secreted by the plant in response to attack by parasites such as insects, fungi, viruses, etc and which display activity against these parasites, and (b) chemicals which encourage colonisation of nitrogen-fixing bacteria on the roots of legumes. The biological functions in plants of the lignans and coumestans is not generally understood
The different types of phyto-oestrogens are as follows.
Type 1 Phyto-oestrogensxe2x80x94(Isoflavones)
Isoflavones appear to be widely distributed in the plant kingdom and over 700 different isoflavones are described. However, the isoflavones which display oestrogenic activity belong to a small sub-group and are restricted almost exclusively to the Leguminosae family. The known oestrogenic isoflavones are daidzein, formononetin, genistein and biochanin A. In common human foodstuffs such as soya, chickpeas, lentils and beans, the total levels of the oestrogenic isoflavones range between about 40 and 300 mg per 100 g dry weight.
In the raw plant material, isoflavones occur principally as glycosides. Following ingestion by man and animals, the glycoside moiety is hydrolysed free by a combination of gastric acid hydrolysis and fermentation by intestinal bacteria. Some of the isoflavones in the aglucone form are absorbed directly and circulate in the blood, while the remainder are metabolised by intestinal fermentation to a variety of compounds which are also absorbed. The absorbed isoflavones and their metabolites appear to undergo little or no further metabolism in the body, being readily transported in the bloodstream, and ultimately being excreted in the urine.
Type 2 Phyto-oestrogens (Lignans)
Lignans are widely distributed in the plant kingdom. Over one hundred lignans are described and they are reported in common human foodstuffs such as cereals, fruits and vegetables. Oilseeds such as flax (linseed) have the highest known levels at 20-60 mg/100 g dry weight, while cereals and legumes have much lower levels at 0.3-0.6 mg/100 g, and vegetables even lower levels at 0.1-0.2 mg/190 g. The most common lignan described is metairesinol. Dietary lignans also appear to be metabolised fairly efficiently within the gut by bacterial fermentation, yielding metabolites such as enterodiol and enterolactone which are absorbed into the bloodstream and excreted in the urine.
Type 3 Phyto-oestrogens (Coumestans)
Compared to isoflavones and lignans, oestrogenic coumestans appear to have a relatively restricted distribution in plants and generally occur at much lower levels. Alfalfa, ladino clover and some other fodder crops such as barrel medic may have significant levels and have been reported to cause reproductive dysfunction in grazing animals. In the human diet, the important sources of coumestans are sprouts of soya and alfalfa where levels up to 7 mg/100 g dry weight are reported. Whole soyabeans and other common foodstuff legumes contain levels of approx. 0.12 mg/100 g dry weight and most of that is concentrated in the seed hull which commonly is removed in the preparation of human foodstuffs.
Type 4 Phyto-oestrogens (Oestrogens)
These are compounds closely related to animal oestrogens such as oestrone, oestradiol and oestriol. These have been described in plants such as liquorice, apple, French bean, pomegranate and date palm. Little is known of the metabolism and biological significance of these chemicals in humans and animals.
The full range of biological effects in animals of these dietary phyto-oestrogens has received only recent study. A primary effect appears to be associated with their close structural relationship to naturally-occurring oestrogens which allows the phyto-oestrogens to mimic the effects of the endogenous oestrogens. The known biological effects of phyto-oestrogens can be summarised thus:
In vitro
(a) bind to both cytoplasmic and nuclear membrane (Type II) oestrogen receptors on human tissues;
(b) strongly compete with oestrogens for oestrogen receptors, but only weakly stimulate those receptors;
(c) strongly stimulate the production of sex hormone-binding globulin (SHBG) from human cells;
In vivo
(d) weakly oestrogenic in animals;
(e) competitively-inhibit the response of tissue to oestrogens.
The three major types of phyto-oestrogens appear to act at the cellular level in a similar manner, that is through interaction with cell surface oestrogen receptors. In the body, naturally-occurring oestrogens circulating in the blood largely exert their activity by interaction with oestrogen receptors on cell surfaces; such interactions then triggering a particular biological function of that particular cell. Phyto-oestrogens are able to bind to those oestrogen receptors because the structure of these compounds so closely resembles the endogenous oestrogens, but unlike the animal oestrogens, phyto-oestrogens only weakly activate the oestrogen receptor.
As a result of phyto-oestrogens and endogenous oestrogens competing for the oestrogen-binding sites on cells, the more weakly oestrogenic phyto-oestrogens can be considered to have an anti-oestrogenic effect. This phenomenon is known as competitive-inhibition, by which is meant that the biological effect of an active substance is impaired by the competitive binding to a target receptor of a similar but less active compound.
Thus a primary biological effect of phyto-oestrogens is held to be competitive inhibition of endogenous oestrogens. However, another more direct effect is the stimulation of synthesis of SHBG in the liver, as occurs with orally administered synthetic steroidal oestrogens. High levels of dietary phyto-oestrogens are thought to be responsible for the higher SHBG levels seen in vegetarians and in cultures maintaining traditional (high legume-containing) diets.
At high levels, dietary phyto-oestrogens can have profound physiological effects. An example of this is sheep and cattle grazing pastures containing a high proportion of subterranean clover or red clover which can contain levels of phyto-oestrogens as high as 5% of the dry weight of the plant. As a result of the competitively-inhibitory effect of the dietary phyto-oestrogens on endogenous oestrogen function in the hypothalamus, male and female sheep and cows can develop androgenic symptoms.
Such high dietary levels of phyto-oestrogens, however, are rare. It is far more common that most animal and human diets contain low to moderate levels of phyto-oestrogens, and there is growing epidemiological evidence that such levels have a beneficial effect on human health.
In most traditional human diets in developing countries, the principal phyto-oestrogens consumed are isoflavones because of the generally high reliance on legumes (also known as pulses) as a source of protein. The general consumption rates (g/day/person) for legumes for different regions currently are approximately: Japan (50-90), India (40-80), South America (30-70), North Africa (40-50), Central/Southern Africa (20-50) and Southern Mediterranean (30-60). Legumes also are a source of lignans and, to a much lesser extent, coumestans, and the additional cereal and vegetables in the diet would also boost the lignan intake. However, the isoflavone intake in these traditional cultures with high legume consumption would typically be much in excess of either lignan or coumestan intake.
The major types of legumes used in traditional diets include soya, chickpeas, lentils, ground nuts, beans (e.g. broad, haricot, kidney, lima, navy), and grams (bengal, horse and green).
In Western, developed countries, the daily intake of dietary phyto-oestrogens generally is negligible to low. In Western Europe, North America and Australasia, legumes were a major source of protein for the majority of the populations up to the end of the 19th century. From that time, legume consumption has declined significantly, being replaced in the diet with protein of animal origin. Average legume consumption in these regions currently is between 5-15 g/day/person with a significant proportion of the population ingesting little to no legumes or other phyto-oestrogen containing foods on a regular basis. Moreover, the types of legumes consumed in these regions (e.g. garden peas, French beans) have a typically lower isoflavone content than legumes such as soya and chick peas.
Based on typical consumption rates and types of foodstuffs consumed, the typical phyto-oestrogen intake (mg/day) for different regions can be calculated approximately as
Thus it can be seen that regions which have maintained traditional diets have a higher average daily intake of phyto-oestrogens, particularly isoflavones, compared to western countries. People in communities such as Japan or developing countries with high legume intake excrete substantially higher phyto-oestrogen metabolites in their urine compared to people in Western countries. Within the latter, vegetarians also excrete higher phyto-oestrogen metabolite levels than do those consuming a more typical, omnivorous Western diet.
The presence of relatively large amounts of phyto-oestrogen metabolites in urine serves to highlight their potential biological significance. It has been shown that total urinary excretion of isoflavones and their active metabolites in people consuming moderate amounts of legumes is greatly in excess (up to 10,000xc3x97) of steroidal oestrogen levels. So that while the oestrogenicity of isoflavones to oestrogen receptors is only about 1% that of endogenous oestrogens, this weaker effect is off-set by the much higher blood levels of the isoflavones.
It is known that legumes have formed an important part of the human diet over the past 20,000-30,000 years. It therefore follows that human metabolism has evolved over at least this period in the presence of relatively large levels of dietary phyto-oestrogens, particularly isoflavones. Given the known biological effects of phyto-oestrogens, it also follows that endogenous oestrogen metabolism and function has evolved in the face of significant competitive inhibiting effects of phyto-oestrogens. It has been speculated that the presence of significant dietary levels of phyto-oestrogens in recent human evolution has led to a degree of adaption by tissues responsive to reproductive hormones to these dietary components. That is, both the rate of production and/or the function of endogenous oestrogens may be either dependent upon or influenced by the presence of phyto-oestrogens in the body. It follows therefore that a relative deficiency of dietary phyto-oestrogens could be expected to lead to an imbalance of endogenous oestrogen metabolism.
There is increasing interest in the likely contribution of a relative deficiency of dietary phyto-oestrogens to the development of the so-called xe2x80x9cWester diseasesxe2x80x9d, especially cancer of the breast, benign (cystic) breast disease, cancer of the uterus, cancer of the prostate, cancer of the bowel, pre-menstrual syndrome, menopausal syndrome, and atherosclerosis. All of these diseases are associated to a greater or lesser extent to oestrogen metabolism, and oestrogen function is either known or is suspected to play a role in their aetiology and/or pathogenesis.
Each of these diseases occurs at much higher incidence in Western, developed countries than it does in developing communities. Moreover, it is thought that in Western communities, the incidences of each have risen over the past century. It is also generally held, that of all the environmental factors likely to be contributing to this phenomenon, diet is the principal factor. Of those dietary components with the potential to influence the aetiology of oestrogen-related disease, there is a growing awareness that phyto-oestrogens may have important potential.
The beneficial effects of phyto-oestrogens on human health are thought to derive from at least two principal function, those being (i) competitive-inhibition of the function of endogenous oestrogens, and (ii) the stimulation of production of SHBG. SHBG plays an important role in primates in binding and transporting the reproductive hormones (oestrogens, androgens) in blood so that the availability of reproductive hormones is regulated to a large degree by SHBG levels. Higher SHBG levels are considered beneficial in leading to a reduction in both blood levels of unbound (and unregulated) reproductive hormones and metabolic clearance rates of the hormones. Although isoflavones are potent stimulators of SHBG synthesis, they only weakly bind to SHBG, so that the increased SHBG levels resulting from the dietary isoflavones are largely available for binding to endogenous oestrogens.
In terms of directly identifying the beneficial effects of phyto-oestrogens in amelioration of any or all of the xe2x80x9cWestern diseasesxe2x80x9d, there are only two examples. In one example, the diets of women, with menopausal syndrome were supplemented with foodstuffs (soya, linseed, red clover) high in phyto-oestrogens, and an alleviation of menopausal symptoms to an extent similar to that obtained with replacement therapy with synthetic oestrogens was achieved; that effect was ascribed to the phyto-oestrogen content of the supplement. In the other example, legumes such as soya and various pulses have been shown to have a hypocholesterolaemic effect in humans; this effect has not been ascribed to phyto-oestrogens, although purified isoflavones do have a hypocholesterolaemic effect in animals with artificially-induced hypercholesterolaemia.
In summary, it could reasonably be deduced that the inclusion of greater levels of foodstuffs high in phyto-oestrogens in the standard diets of men and women in developed countries could be expected to redress a general imbalance of endogenous reproductive hormone metabolism, thereby reducing the predisposition of those communities to the above diseases. While there are various types of phyto-oestrogens which may be suitable to this end, the large discrepancy in isoflavone consumption between communities with Western and traditional diets suggest that foodstuffs with high isoflavone content are of prime interest.
However it is unrealistic to expect that public education programmes would readily convert communities in developed countries from a diet where the protein content is predominantly animal-derived, to one where the protein is predominantly legume-derived. Moreover, the legumes which are commonly consumed in developed countries are relatively poor sources of phyto-oestrogens and the general acceptance in the community of less well-known legumes with higher phyto-oestrogen content would be necessarily a slow process. Also, the highly variable levels of phyto-oestrogens in foodstuffs relating to plant strain type, degree of plant maturity, and climatic and other environmental conditions suggests that the supply of an assured amount of phyto-oestrogens through the use of whole foodstuffs may be difficult.
An alternative strategy is to make available either (i) phyto-oestrogens in a purified form, or (ii) foodstuffs which are enriched for phyto-oestrogens. In this way, the phyto-oestrogen could be added to the diet in a convenient form as a supplement without requiring any substantive change to the diet.
The present invention concerns a health supplement specifically enriched for isoflavones selected from genistein, daidzein, formononetin and biochanin A, or their natural glycoside form, or their analogues, in sufficient amounts to improve the health of a human.
Preferably the supplement contains an excipient, a diluent, a carrier or the like, or else the supplement is mixed with food or can be consumed directly. It is also preferred that foodstuffs, are readily available, have no known toxic components, and are rich sources of isoflavones; such foodstuffs preferably being red clover or soya. It is also preferred that the ratio of genistein and/or it methylated derivative biochanin A to daidzein and/or its methylated derivative formononetin is between 1:2 to 2:1. Other plant components with oestrogenic activity including lignans, coumestans and flavones may also be present in the extract, but it is held that these are of secondary importance to the predominant isoflavones. The term phyto-oestrogens is used hereafter to indicate a predominance of isoflavones with lesser amounts of lignans, coumestans and flavones.
The invention also concerns a method of improving the health of a human by administering to the human a sufficient amount of phyto-oestrogen. Ideally, the phyto-oestrogen is administered regularly on a daily basis over a sufficient period such as at least a month. The health conditions which may be prevented or ameliorated include cancer of the breast, cancer of the prostate, cancer of the uterus, cancer of the bowel, benign (or cystic) breast disease, pre-menstrual syndrome (also known as pre-menstrual tension), or adverse symptoms associated with menopause in women. The method and supplement in accordance with the invention also improves the health of a human having elevated levels of blood cholesterol. The product also is useful in avoiding or ameliorating cancer in persons. The symptoms produced by these conditions and the general well-being is also improved by the use of these supplements.
The phyto-oestrogen in accordance with the invention may be obtained from a number of different sources. Preferably the phyto-oestrogens are extracted from a clover such as red clover or subterranean clover or from soya which contain high levels of phyto-oestrogens.
However, any source rich in phyto-oestrogens may be used instead, if desired.
Various different isoflavones have been identified from these sourcesxe2x80x94they are principally genistein, biochanin A, daidzein, formononetin and glycitein. In plants these compounds occur principally in a glycoside form bound to sugars such as glucose, with smaller amounts present as the aglucone forms. The formulae of the isoflavones are:
The structure of biochanin A is the same as for genistein but with a 4xe2x80x2-methoxy group, and similarly formononetin has the same structure as daidzein, but with a 4xe2x80x2-methoxy group.
Following ingestion by humans, the glycosidic isoflavones are hydrolysed to the aglucone form and biochanin A and formononetin are demethylated by bacterial fermentation to genistein and daidzein respectively. A small proportion of these free isoflavones are absorbed directly from the bowel and circulate in the blood. The bulk of the isoflavones, however, remain in the bowel and undergo fermentation to form various metabolites which also are absorbed into the bloodstream. The principal metabolites which have been identified are equol and O-desmethylangolensin.
In vitro and in vivo studies have indicated that genistein, biochanin A, equol, daidzein, formononetin all have oestrogenic activity in descending order. O-desmethylangolensin is only very weakly oestrogenic and glycitein is non-oestrogenic.
In animal and in vitro studies, genistein has been shown to have greater oestrogenic/anti-oestrogenic activity and SHBG-stimulating capacity than the other isoflavones or their metabolites (approximately 10 times that of daidzein and formononetin). However, the full range of biological effects of the different isoflavones have yet to be fully determined, and in particular their relative efficacies in the different biological effects such as oestrogenicity, hypocholesterolaemia, anti-angiogenesis, anti-oxidation, anti-carcinogenesis for example are not yet fully known.
It is thought that because the methyl forms (biochanin A and formononetin) ultimately are largely demethylated to their principals, genistein and daidzein, with improved biological efficacy, then it is unimportant whether the isoflavones are present in the claimed product in the methylated or demethylated forms.
Given that the relative biological importance of the two isoflavone groups (being genistein and daidzein) to human health remains unclear, and that each might indeed have different importance, plus the fact that both isoflavones are present in the diet in approximately equal proportions, then it is prudent that both isoflavones be present in the claimed product in approximately equal proportions.
Any leguminous plants such as detailed here could be used as sources of phyto-oestrogens (principally isoflavones with lesser amounts of lignans and coumestans): Indian liquorice (Abrus precatorius); various species of Acacia spp. including, A. aneura, A. cibaria, A. longifolia, and A. oswaldii; ground nut (Apio tuberosa); ground pea (Arachis hypogea); milk vetch (Astragalus edulis); marama bean (Bauhinia esculenta); sword bean (Cajanus cajan indicus); jack bean (Canavalia ensiformis); sword bean (Canavalia gladiata); seaside sword bean (Canavalia rosea); various Cassia spp. including C. floribunda, C. laevigata, and C. occidentalis; carobbean (Ceratonia siliqua); chick pea (Cicer arietinum); yebnut (Coideauxia edulis); various Crotalaria spp. including C. laburnifolia, and C. pallida; cluster bean (Cyamopsis psoralioides); tallow tree (Detariaum senegalense); sword bean (Entada scandens); balu (Erythrina edulis); soyabean (Clycine max;) inga (Ingaedulis); Polynesian chestnut (Inocarpus fagifer); hyacinth bean (Lablab purpureus); grass pea or Indian vetch (Lathyrus sativus); cyprus vetch (Lathyrus ochrus); lentil (Lens culinaris); jumping bean (Leucaenal eucocephala); various Lupinus spp. including L. albus, L. luteus, L. angustifolium, L. mutabilis, and L. cosentinii; ground bean (Macotylma geocarpa); horse gram (Macrotyloma uniflorum); alfalfa (Medicago sativa); velvet bean (Mucuna pruriens); yam beans (Pachyrhyzuz erosus, P. tuberosus); African locust bean (Parkia clappertoniana); Parkia speciosa; oil bean tree (Peniaclethra macrophylla); various Phaseolus spp. including P. acutifolius, P. vulgaris, P. luntus, P. coccineus, P. adenathus, P. angulris, P. aureus, P. calcarutus, P. mungo, and P. polystachyus; garden pea (Pisum sativum); djenko bean (Pithecolobium lobatum); mesquite (various Prosopis spp.); goa bean (Psophocarpus scandens, P. tetragonolobus); various Psoralea spp.; Sesbania bispinosa; yam bean (Sphenostylis stenocarpa); tamarind (Tamarindus indica); fenugreek (Trigonella foenum-graecum); vetches (various Vivia spp. including V. sativa, V. atropurpurea, V. ervilia, and V. monantha); broad bean (Vicia faba); black gram (Vigna mungo); various Vigna spp. including V. radiata, V. aconitifolia, V. adanatha, V. angularus, V. tribolata, V. umbelata, and V. unguiculata, and, earth pea (Voandzeia subterranea).
The ideal sources of phyto-oestrogens for preparation of a supplement in accordance with the invention are preferably those which (i) are readily available, (ii) are relatively inexpensive, (iii) are readily and economically processed so as to yield the extract, (iv) have a high isoflavone content so as to provide high yields, and (v) have no known toxic components requiring selective removal or inactivation.
Certain clovers, such as red clover (T. pretense) and subterranean clover (T. subterranean) are the preferred sources. On a dry weight basis, these clovers contain the highest amounts of oestrogenic isoflavones of all legumes tested to date with levels of 3-5 g % (T. subterranean) and 1-3 g % (T. pratense). In comparison, soya flour has a level of 0.15-0.30 g %, lentils (0.08-12 g %), chick peas (0.07-0.13 g %), and garden peas (0.02-0.03 g %). Thus it can be seen that clovers contain approximately at least 10-30 times by weight the isoflavone content of other commonly available, human leguminous foodstuffs meaning that for manufacturing purposes, the yield of isoflavones per unit weight of plant material is many times greater from clover than from other legumes.
Red clover and subterranean clover also are common fodder crops and are readily grown and are widely available. Clovers also are comparatively cheaper ($200/tonne) than crops such as soya and lentils ($500/tonne).
With clovers, the isoflavones are recovered from the leaf rather than from the seed in the case of soya, beans, nuts and grams. This provides a substantially higher yield of isoflavones per unit area of pasture for clovers compared to other legumes because of the greater leaf matter compared to seed matter recovered per plant.
Clovers also have an extended growing season, and faster growth rates compared to those legumes such as soya, lentils or chick peas where the seed is the end-product. Clover can be cropped for its leaf content repeatedly over a single, growing season. An additional benefit of this is that as phyto-alexins, the isoflavone content increases in response to the stress of cropping.
Thus it can be seen that in clovers versus other legumes provide a combination of (a) higher isoflavone content per dry weight of plant, (b) a higher yield of dry matter containing isoflavones per plant, and (c) a higher yield of dry matter per hectare.
An additional feature of clovers is that there are wide varieties of cultivars with widely differing isoflavone levels and types. This allows blending of different cultivars to achieve the desired ratio of the different isoflavones, although it is equally possible to use a single cultivar which provides the desired ratio.
Other legumes such as soyabean flour may be used for enrichment of phyto-oestrogens but the substantially poorer (approx. 10%) yield of isoflavones compared to clovers means that the manufacturing costs are substantially greater and there is substantially greater amounts of waste products which requires disposal or further treatment for re-use as a foodstuff. An alternative, however, to the use of whole soya for this purpose, is to use the hull and hypocotyl (or germ) of the whole soyabean. The hull and hypocotyl represent only a small proportion by weight (8% and 2% respectively) of the intact bean. However, the coumestrol content of soya is concentrated in the hull, and the daidzein content of soya is concentrated in the hypocotyl. The two cotyledons which comprise the bulk of the soyabean (90% by weight) contain the bulk of the genistein content of soya. During standard processing of soyabeans, the hulls being a fibrous component with little or no perceived nutritional value normally are separated and removed by physical means. The hypocotyls become separated following the splitting of the cotyledons, and while these currently generally are not deliberately isolated, they may be separated and isolated by passing the disturbed soyabeans over a sieve of sufficient pore size to selectively remove the small hypocotyl. The hypocotyl contains approx. 1.0-1.5 g % isoflavones (95% daidzein, 5% genistein). The raw hypocotyl and hull material can be ground or milled to produce, for example, a dry powder or flour which then could be either blended or used separately as a dietary supplement in a variety of ways including, for example, as a powder, in a liquid form, in a granulated form, in a tablet or encapsulated form, or added to other prepared foodstuffs. Alternatively, it could be further processed to yield an enriched extract of phyto-oestrogens. Either or both of these materials also could be added to other leguminous material such as clover to provide the invention.
In plants, the oestrogenic isoflavones are restricted principally to the leaf, fruit and root; the stem and petiole contain very little. With soya and other common human legume foodstuff crops, the leaves are rarely regarded as foodstuff; indeed with these crops, the plants normally are allowed to die and dry out before the seed crop is harvested. Nevertheless, the fresh leaves of these crops could be regarded as a source of phyto-oestrogens for the invention although the much lower isoflavone content of the leaves of these crops compared to clovers, plus their generally slow growth compared to clovers, suggests that they would not be a preferred source of large-scale isoflavone enrichment.
To provide a similar amount of isoflavone to that contained in most traditional legume-rich diets (50-100 mg oestrogenic isoflavones/day) would require an average daily consumption of 3-6 g dry weight or 15-30 g wet weight of specially selected cultivars of clover with particularly high isoflavone levels. Clover grasses generally are not eaten by humans, except to a limited extent as sprouts of some of the pleasanter tasting varieties. Isoflavones are intensely astringent and are responsible in large part for the bitter taste of legumes. Thus the types of bean sprouts, clover sprouts and alfalfa sprouts generally available have been selected on the basis of cultivar and of age for pleasant taste, and in so doing inadvertently have been selected for low isoflavone content. Of the sprouts currently available in Western countries for human consumption, between approx. 100-250 g would need to be consumed daily to provide a dosage of 50-100 mg isoflavones. Certainly clovers and other legume sprouts are not generally eaten in such sufficient quantities by humans to obtain the advantages of the present invention
The invention also concerns formulations containing the phyto-oestrogens discussed above together with a dietary suitable excipient, diluent, carrier, or with a food. Ideally the formulation is in the form of a pill, tablet, capsule, or similar dosage form.
The formulations may be a variety of kinds, such as nutritional supplements, pharmaceutical preparations, vitamin supplements, food additives or foods supplemented with the specified active phyto-oestrogens of the invention, liquid or solid preparations, including drinks, sterile injectable solutions, tablets, coated tablets, capsules, powders, drops, suspensions, or syrups, ointments, lotions, creams, pastes, gels, or the like. The formulations may be in convenient dosage forms, and may also include other active ingredients, and/or may contain conventional excipients, carriers and diluents. The inclusion of the subject phyto-oestrogens in herbal remedies and treatments is also a preferred part of the invention.
The invention is also directed to the amelioration, prevention, or of various conditions responsive to treatment with the phyto-oestrogen substances of the invention. The preferred amounts to be administered to the human fall within 20-200 mg on a daily basis. More preferably the dosage is from 50-150 mg on a daily basis, and most preferably at a dosage of about 100 mg. If desired greater dosages can be administered for therapeutic reasons. In contrast to prior practices such high dosages were not possible. For example, dosages of up to or greater than 1000 mg may be suitable to treat some conditions. In order to obtain the benefits of the invention, the treatment with the isoflavones should continue for a considerable period, ideally for at least a month, and ideally continuously for the whole period for which the health improvement advantages should accrue.
The product according to the present invention yields a constant and accurately known amount of isoflavones. The product is also ideally a natural product, which has advantages for consumer acceptance, and in accordance with the supposed theory behind the invention may very possibly be one of the main causes for its beneficial effects. Whole legumes have a widely variable isoflavone content due to two main causes: the type of legume and the environmental effect. The type of legume typically has a wide range of isoflavone content. The miligram of isoflavone per hundred gram of whole foodstuff (dry weight) is given in the following table:
Thus common leguminous foodstuffs consumed in Western countries (broad beans, garden peas etc) have relatively low oestrogenic isoflavone content and exceptionally large amounts of these would need to be consumed daily to approximate those isoflavone levels consumed in traditional diets. Most Western cultures do not traditionally eat legumes with high isoflavone contents, and those soya products (milk, tofu etc) which are becoming increasingly popular in Western countries, also have relatively low isoflavone levels compared to whole soya, indicating that relatively large amounts of these would need to be consumed on a regular basis to deliver the required isoflavone levels.
The enviromental effect arises because the isoflavone levels in any species of plant depend greatly on the age of the plant, the climatic conditions where it is grown, the fertiliser and so forth. Therefore constant and consistant dosage is very difficult with ordinary whole foodstuffs. The accurately determined quality and quantity of the active isoflavones in the product, and its easy consumability when compared with the almost impossible task of eating huge amounts of often practically inedible foods, is therefore an import feature of the invention for preventing and helping in overcoming various health problems.
Among the various health problems, the treatment or prevention of high blood cholesterol levels, and the treatment of PMS and menopausal symptoms is especially important. The product of the invention modulates the production and/or function of endogenous sex hormones in humans to modify or produce health improving effects, including the following: (i) lowered levels of various blood lipoproteins including, for instance, low-density and very-low-density cholesterol leading to reduced risk of development of atherosclerosis; (ii) reduced risk of development of cancer of the prostate; (iii) reduced risk of cancer of the breast; (iv) reduced risk of development of cancer of the uterus; (v) reduced risk of development of cancer of the large bowel; (vi) reduced risk of development of the syndrome in women commonly referred to pre-menstrual syndrome (PMS), which includes pre-menstrual tension (PMT); (vii) reduced risk of development of many untoward symptoms (including dry vagina, peripheral flushing, depression etc) commonly associated in women with menopause; and for treating benign breast disease in women (benign or cystic breast disease associated with non-malignant swelling and tenderness of breast tissue). The invention therefore is directed to a method for the prophylaxis or treatment of a human, to combat conditions associated with phyto-oestrogen deficiency, which comprises administering to the human an effective amount of phyto-oestrogen principally isoflavone but which might also include relatively smaller amounts of lignans and coumestans, ideally in a concentrated form, wherein the isoflavones include genistein, and/or biochanin A, and/or daidzein, and/or formononetin.
Cancer of the breast generally is considered to be associated with oestrogenic dysfunction. Breast cancer cells may display more oestrogen receptors than normal breast cells and stimulation of these receptors by endogenous oestrogens is thought to be a prime source of stimulation of their malignant growth. Currently synthetic anti-oestrogens are being used to prevent or treat the growth of malignant breast cells. Isoflavones are potent anti-oestrogens that could be expected to help prevent or to successfully treat breast cancer. It has been reported that the risk of breast cancer in western societies is indirectly proportional to the level of phyto-oestrogens in the diet and to the amounts of phyto-oestrogen metabolites excreted in the urine.
Cancer of the prostate generally is considered to be associated with sex hormone dysfunction and the growth of prostatic cancer cells is influenced by oestrogens and androgens. The incidence of prostatic cancer is low in communities with high legume intake and, conversely, is high in Western socieites. Phyto-oestrogens are though to protect from development of prostatic cancer. One mechanism may be the effect of phyto-oestrogens on lowering the proportion of unbound:bound reproductive hormones in the blood. However, there is other evidence to suggest that phyti-oestrogens, particularly isoflavones, can have a direct influence on certain cellular enzymes within prostatic cells.
Pre-menstrual syndrome has uncertain aetiology and pathogenesis, although most certainly is associated with reproductive hormone dysfunction. It also is a syndrome which has reportedly lower incidence in communities maintaining traditional high-legume diets. It is proposed that phyto-oestrogens will alleviate this condition by restoring balance to oestrogen metabolism.
Menopausal syndrome is associated with changes in the oestrogen profile in the body with advancing age. Adverse clinical symptoms may be treated with oestrogen replacement therapy. There is evidence that foodstuffs high in phyto-oestrogens are a suitable alternative to synthetic hormones in this respect, producing alleviation of adverse clinical symptoms. Again, it is proposed that phyto-oestrogens will function by restoring balance to oestrogen metabolism.
Benign (or cystic) breast disease has unknown aetiology. However, its association in women with certain stages of the menstrual cycle is strongly suggestive of oestrogen dysfunction. There currently is no successful treatment of this condition. Phyto-oestrogens are proposed to successfully treat this condition by restoring balance to oestrogen metabolism. Atherosclerosis is associated with cholesterol metabolism which in turn is associated closely with oestrogen metabolism. The generally higher incidence of atherosclerosis in young men versus young women, the rising incidence in women following menopause, and the lower incidence in post-menopausal women receiving oestrogen replacement therapy, all point to the moderating influence of oestrogens on cholesterol metabolism. A prime effect of oestrogens on cholesterol metabolism is stimulation of the liver to process cholesterol, particularly the highly atherogenic low-density lipoproteins and very low-density lipoproteins, into bile salts. It is proposed that phyto-oestrogens have an important hypocholesterolaemic effect in humans. There may be a variety of mechanisms involved, but one may be the stimulation by phyto-oestrogens of cholesterol catabolism by the liver.

Health supplements containing phyto-oestrogens, analogues or metabolites thereof


A system for alleviating occipital neuralgia. The system has a needle probe having at least one needle. The at least one needle has a proximal end, a distal end, and a needle lumen therebetween, the needle configured for insertion proximate to a location of the occipital nerve. A cooling fluid supply lumen extends distally within the needle lumen to a distal portion of the needle lumen. A cooling fluid source is coupled to the cooling fluid supply lumen to direct cooling fluid flow into the needle lumen. A controller that has at least one processor configured implements an occipital neuralgia treatment algorithm for controlling the cooling fluid source so that liquid from the cooling flow vaporizes within the needle lumen to provide a treatment phase to location of the occipital nerve such that the occipital neuralgia is mitigated.

Occipital neuralgia s a medical condition characterized by chronic pain in the upper neck, back of the head and behind the eyes. Often, occipital nerualgia cuases a distinct type of headache characterized by piercing, throbbing, or electric-shock-like chronic pain in the upper neck, back of the head, and behind the ears, usually on one side of the head. Typically, the pain of occipital neuralgia begins in the neck and then spreads upwards. Some individuals will also experience pain in the scalp, forehead, and behind the eyes. Their scalp may also be tender to the touch, and their eyes especially sensitive to light. The location of pain is related to the areas supplied by the greater and lesser occipital nerves, which run from the area where the spinal column meets the neck, up to the scalp at the back of the head. The pain is caused by irritation or injury to the nerves, which can be the result of trauma to the back of the head, pinching of the nerves by overly tight neck muscles, compression of the nerve as it leaves the spine due to osteoarthritis, or tumors or other types of lesions in the neck. Localized inflammation or infection, gout, diabetes, blood vessel inflammation (vasculitis), and frequent lengthy periods of keeping the head in a downward and forward position are also associated with occipital neuralgia. In many cases, however, no cause can be found. A positive response (relief from pain) after an anesthetic nerve block will confirm the diagnosis.
Treatment is generally symptomatic and includes massage and rest. In some cases, antidepressants may be used when the pain is particularly severe. Other treatments may include local nerve blocks and injections of steroids directly into the affected area.
Occipital neuralgia is not a life-threatening condition. Many individuals will improve with therapy involving heat, rest, anti-inflammatory medications, and muscle relaxants. Recovery is usually complete after the bout of pain has ended and the nerve damage repaired or lessened.

Methods and systems for treatment of occipital neuralgia


The present invention provides compositions and methods of use comprising a chimeric dengue virus E glycoprotein comprising a dengue virus E glycoprotein backbone, which comprises amino acid substitutions that introduce a dengue virus E glycoprotein domain I and domain II hinge region from a dengue virus serotype that is different from the dengue virus serotype of the dengue virus E glycoprotein backbone.

Dengue is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 50 countries. Even though infected individuals develop potent and long-lasting serotype-specific neutralizing antibodies (Abs), the epitopes engaged by human neutralizing Abs have not been identified. Here, we demonstrate that the dengue virus (DENV)-specific serum Ab response in humans consists of a large fraction of cross-reactive, poorly neutralizing Abs and a small fraction of serotype-specific, potently inhibitory Abs. Although many mouse-generated, strongly neutralizing monoclonal antibodies (MAbs) recognize epitopes that are present on recombinant DENV envelope (E) proteins, unexpectedly, the majority of neutralizing Abs in human immune sera bound to intact virions but not to the ectodomain of purified soluble E proteins. These conclusions with polyclonal Abs were confirmed with newly generated human MAbs derived from DENV-immune individuals. Two of three strongly neutralizing human MAbs bound to E protein epitopes that were preserved on the virion but not on recombinant E (rE) protein. It is proposed that humans produce Abs that neutralize DENV infection by binding a complex, quaternary structure epitope that is expressed only when E proteins are assembled on a virus particle. Mapping studies indicate that this epitope has a footprint that spans adjacent E protein dimers and includes residues at the hinge between domains I and II of E protein. These results have significant implications for the DENV Ab and vaccine field.

Chimeric dengue virus E glycoproteins comprising mutant domain I and domain II hinge regions

The University of North Carolina at Chapel Hill


A transparent or substantially transparent formable and/or flexible component for use as an outer protective element in an electronic or opto electronic device including at least one electrically active organic layer, which component is a composite structure comprising a layer of glass of a thickness less than or equal to 200 microns and a layer of plastic.

This invention relates to flexible substrates for organic devices and particularly but not exclusively to organic light emitting devices (OLEDs) and to OLEDs fabricated on flexible substrates.
Organic light-emitting devices such as described in U.S. Pat. No. 5,247,190 or in U.S. Pat. No. 4,539,507, the contents of which are incorporated herein by reference, have great potential for use in various display applications. According to one method, an OLED is fabricated by coating a glass or plastic substrate with a transparent first electrode (anode) such as indium tin oxide (ITO). At least one layer of a thin film of an electroluminescent organic material is then deposited prior to a final layer which is a film of a second electrode (cathode) which is typically a metal or alloy.
The electrode and organic layers used in OLEDs are typically very thin; normally of the order of a few 100 nm and typically around 100 nm and can be flexed without a great risk of damage to the device structure and functioning of the device. By using thin substrates of glass or transparent plastic, formable and/or flexible light sources and displays can be made. For this purpose substrates can be at most a few 100 xcexcm thick.
In order to fabricate OLEDs which have good operating and shelf life it is of utmost importance to protect the active layers of the device, i.e. the electrode and organic layers, from the ingress of ambient species which can react with the active layer and deteriorate device performance, particularly oxygen and moisture. Typically, but not necessarily, an OLED emits light only from one side and this is typically through the transparent substrate and anode. The cathode is typically opaque and is made of a metal or alloy. This opaque side is relatively easy to encapsulate against the ingress of ambient reactive species as, for example, pinhole-free metal foils or metallised plastic foils can be used by, for example, lamination to the cathode.
For OLEDs fabricated on glass substrates the glass itself provides an excellent barrier against the ingress of oxygen and moisture. However, for OLEDs fabricated on transparent plastic foils it is extremely difficult to encapsulate the transparent side against the ingress of ambient reactive species. The oxygen and water permeabilities of even the most impermeable transparent plastic substrates (thin films) presently available are too high to be sufficient as a barrier for long life OLED devices. A simple estimate for this is given for example in K. Pichler, Phil. Trans. R. Soc. Lond. A (1997), Vol. 355, pp 829-842. This situation can be greatly improved by the conductive transparent coating itself, typically an inorganic conductive oxide such as indium tin oxide (ITO). Such ITO coatings on the thin plastic substrates can be very good barriers against the ingress of oxygen and water from outside into the device, as long as the ITO coatings are pinhole-free and defect free. However, these thin ITO (or other conductive oxide coatings) deposited onto thin flexible plastic substrates are prone to xe2x80x9ccrackingxe2x80x9d if the substrates are not handled with the greatest care. The occurrence of such cracks in the ITO coating creates highly efficient diffusion channels for the ingress of ambient reactive species, just as pinholes in the coating would do. In addition to that, such cracks in the ITO coating may also result in an undesired deterioration of the surface flatness of the coating. This requirement to avoid cracking of the ITO coating puts severe constraints on the handling of the substrates and devices and hence the manufacturing process.
Alternatively, the use as an OLED substrate of thin formable and/or flexible glass with thicknesses of less than 200 xcexcm is possible and even only 30 xcexcm thick flexible glass, which is available commercially, is impermeable to oxygen and water and thus provides excellent barrier properties together with high transparency. Such thin glass is currently available from, for example, DESAG AG, Germany. However, such thin glass, although of a composition and specially manufactured to reduce brittleness, is still extremely difficult to handle and can very easily break if not handled with the greatest care. This puts severe limitations on the use of thin flexible glass as substrates for OLEDs due to the difficulty of manufacturing.
It is an aim of the present invention to provide an improved substrate for an electronic or optoelectronic device including at least one electrically active organic layer, which avoids or at least reduces the problems of the prior art.
According to one aspect of the present invention there is provided a transparent or substantially transparent formable and/or flexible component for use as an outer protective element in such an organic device, which component is a composite structure comprising a layer of glass of a thickness xe2x89xa6200 xcexcm and a layer of plastic. In this connection the formability of the component allows it to deviate from full planarity by bending and/or twisting so it can adapt to the shape or form of some other object. Its flexibility allows it to be bent without detrimentally affecting its barrier properties.
The invention is particularly but not exclusively concerned with an organic light-emitting device. Such a device comprises a first charge injecting electrode for injecting charge carriers of a first type and a second charge injecting electrode for injecting charge carriers of a second type. Between the first and second electrodes is arranged at least one layer of a thin film of an electroluminescent organic material. When an electric field is applied across the device, charge carriers injected into the material by the first and second electrodes recombine and decay radiatively causing light to be emitted from the electroluminescent layer. In the present description, the first electrode is referred to herein as the anode and the second electrode is referred to herein as the cathode.
Other organic devices include thin film transistors (TFTs), diodes, photodiodes, triodes, photovoltaic cells and photocouplers.
The outer protective element can constitute a substrate for the organic device and, as such, can be coated with a transparent electrode layer. That layer would normally be the anode and is preferably of indium tin oxide. In that case, the electrode coating is applied to one surface of the glass layer so that the plastic layer, adjacent the other surface of the glass layer, forms the outer layer of the protective element. As an alternative, the outer protective element constitutes an encapsulation film for a preformed organic light-emitting device.
In order to form the structure in which the outer protective element constitutes a substrate for the organic light-emitting device, the glass layer may be precoated with a transparent electrode layer prior to attachment to the plastic layer, or the transparent electrode layer may be deposited after fabrication of the composite structure. It is also possible to reverse the order of layers in the composite structure so that the plastic layer constitutes the inner layer carrying the electrode layer and the outer layer constitutes the glass layer.
The organic device with the outer protective element can be manufactured in a sequence of integrated steps which include the construction of the composite structure, deposition of the transparent electrode layer, deposition of the or each organic electrically active layer and deposition of the second electrode layer. A batch, semi-continuous or continuous process can be considered for the manufacture of the complete device. A further encapsulation layer on the second electrode layer can be provided.
Various manufacturing techniques are possible in accordance with different embodiments of the present invention.
According to one embodiment, a plastic layer carrying a coating of a first transparent electrode (e.g. ITO) is provided. Then, at least one layer of an electrically active, e.g. electroluminescent, organic material is deposited followed by the second electrode layer. The complete structure is then laminated to the glass layer.
According to another embodiment, the plastic and glass layers are exchanged in the preceding sequence.
According to a further embodiment, the composite structure is prefabricated and is then used as the basis for deposition of the first electrode layer, the at least one layer of an electrically active organic material and the second electrode layer.
If the glass layer is used as the xe2x80x9couter layerxe2x80x9d, that is with the plastic layer adjacent the first electrode layer and within the glass layer, it is important to remove (for example by out-gassing, baking-out or pumping-out) impurities (for example oxygen, water and possible other low molecular weight compounds) which may be present in the substrate.
Preferably, the glass layer is less than 100 xcexcm thick and better still around or less than 50 xcexcm thick.
Preferably, the plastic layer is less than or around 1 mm thick and preferably less than or around 500 xcexcm thick and better still less than or around 200 xcexcm thick.
The glass and plastic layers can be provided in sheet form according to a batch process.
Alternatively, the glass layer can be provided in sheet form and the plastic layer from a continuous roll.
As a further possibility, both glass and plastic layers are from continuous rolls.
The composite structure can be formed by lamination of the glass and plastic layers, e.g. according to a batch process, a continuous roll-to-roll process or a semi-continuous process whereby the plastic layer is a continuous film and the glass layer is in sheet form.
The plastic layer can be of e.g. polyesters, polycarbonate, polyvinylbuterate, polyethylene and substituted polyethylenes, polyhydroxybutyrates, polyhydroxyvinylbutyrates, polyetherimides, polyamides, polyethylenenaphalate, polyamides, polyethers, polysulphones, polyvinylacetylenes, transparent thermoplastics, transparent polybutadienes, polycyanoacrylates, cellulose-based polymers, polyacrylates and polymethacrylates, polyvinylalcohol, polysulphides and polysiloxanes.
It is also possible to use polymers which can be deposited/coated as pre-polymers or pre-compounds and then converted, such as epoxy-resins, polyurethanes, phenol-formaldehyde resins, and melamine-formaldehyde resins.
The lamination of the glass and plastic layers can be with glue/adhesive in between the two layers. In that case, glue can be pre-coated onto one of the two or on both substrates; or supplied during the lamination process, at room or elevated temperature and with or without pressure. UV-cured glues are also suitable.
The plastic layer can be from Aclam(trademark) or other similar plastic sheets which are pre-coated with a heat-seal glue.
Lamination and/or deposition of the plastic layer onto the glass layer can be integrated in the fabrication process of the glass, i.e. glass comes off the fabrication line and is then (still hot or warm or cold) coated with the plastic.
As an alternative to formation by lamination, the plastic layer of the composite is coated onto the glass layer by a batch or continuous process. Coating of the plastic onto the glass can be by dip, spray, solution-spin, solution-blade, meniscus coating, or by coating of a molten plastic onto the glass layer.
That is, it is possible to consider the different situations (i) where plastic exists already as film and is laminated to the glass and (ii) where plastic is not in film form but is coated onto the glass by dip, spray, etc. etc. The pre-polymers mentioned above, for example, are amenable to case (ii). However, also several of the other plastics mentioned above can be coated for case (ii). In this instance the polymers can be coated onto the glass principally by: coating from solution, from a melt or as pre-polymer.
The invention also provides an organic device comprising:
a flexible and/or formable composite structure comprising a layer of glass of thickness xe2x89xa6200 xcexcm and a layer of plastic;
a transparent or substantially transparent first electrode layer overlying the composite structure;
at least one layer of an electrically active organic material overlying the first electrode layer; and
a second electrode layer overlying the layer(s) of organic material.
In one embodiment, the electrically active organic material is electroluminescent.
The composite structure finds use as a substrate/encapsulant not only for OLEDs (small molecule fluorescence (SMF) and (LEP) light emitting polymers) but for other devices including at least one electrically active organic layer e.g. organic photo-detectors, organic solar-cells, thin-film-transistor (TFT) arrays and TFTs for OLEDs. The preferred use is for LEP products such as un-patterned backlights and other light sources or patterned devices such as signs, alpha-numeric displays or dot-matrix and other high-resolution displays. In particular, a preferred light-emitting polymer is a semiconductive conjugated polymer of the type discussed in the earlier referenced US patents.
It is often the case that the problems with handling thin glass arise from picking up glass sheets at the edges where a crack can start very easily. In order to avoid this, one embodiment of the present invention provides that the plastic layer extends beyond the edges of the glass layer in the composite so that the plastic part only can be used to pick up the composite structure. This reduces cracking possibilities and keeps the glass layer untouched.
The plastic xe2x80x9cexcessxe2x80x9d can be all around the glass substrate, at one edge or at any of a number of edges.
In manufacture of an organic light-emitting device, it is usually necessary to subject some or all of the layers to processing steps. For example, if the electroluminescent organic material is a semiconductive conjugated polymer such as poly(phenylene vinylene) (PPV) then the deposition of that layer would normally take place by depositing a precursor to the polymer in a solvent, for example by spin-coating, and then subjecting that layer to a subsequent processing step to convert the precursor to the final polymer. Thus, the underlying composite structure, if present during these processing steps, must be able to withstand the solvents used for spin-coating the precursor layer and the subsequent temperatures used for driving off the solvent and converting the precursor to the polymer. Thus, the plastic layer of the composite structure needs to be of appropriate qualities. For example, if the composite structure is to be subjected to high temperatures, then the glass-transition temperature of the plastic layer should be above those temperatures. For example, a temperature of in excess of 150xc2x0 C. is possible. Moreover, the plastic layer should be resistant to the solvent layers used for the polymers, such as mixed xylene, THF, used for soluble conjugated polymers such as MEH PPV.
The composite structure can comprise more than two layers. For example, the composite structure can comprise a glass layer and two plastic layers.
In particular, a composite structure can comprise a plastic/glass/plastic composite with a coating of ITO on the outer surface of one of the plastic layers. Such a structure would be preferred in a situation where there was a significant difference in the expansion coefficients of the glass layer and plastic layer, such that a bending of the composite structure may take part. This would be prevented if an additional layer was incorporated.
Additional functionality can be incorporated into the plastic layer. For example, the plastic layer can comprise a plastic polariser sheet, a contrast-enhancing filter-laminate, have anti-reflective properties, colour filter properties or colour conversion properties. For example, it would be possible to have a device in which the light emitting layer emits blue light and in which the laminate contains, for example, red or green fluorescent molecules which absorb the blue and re-emit in the red or green. Alternatively or additionally, the plastic layer can be designed to block undesired ambient light and/or have scattering particles so that wave guiding is reduced and the brightness of the device is increased. Where possible, such additional functionalities could be incorporated in the glass layer. Where a third plastic layer is provided in the composite structure, this allows the possibility of two different types of plastic layers, providing the possibility for incorporating different additional functionalities into the different layers.
The present application thus describes transparent substrates and encapsulation films for use in organic devices which avoids or at least reduces the problems of prior art and provides substrates and films which have both good transparency and good barrier properties and which can also be handled without great risk of deteriorating the continuity of the substrate and its barrier properties.